Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells.

The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells.


Cells
Two stable CHO DG44-derived cell lines (clones 1 and 2) expressing a human anti-RhesusD IgG 9 were used in this study. Both clones were established following gene delivery with the piggyBac transposon 10 .

Osmolality measurement
The osmolality of media was measured using a freezing point computerized micro-osmometer (Multi-Osmette™ 2430, Precision Systems, Natick, MA). The osmolality of the peptone-supplemented media was between 310 and 330 mOsm.

Protein production
The concentration of a human recombinant IgG in the culture medium was determined by sandwich ELISA as previously described 11 . The  (table 1).

Cell cultivation
Clones 1 and 2 were routinely cultivated in × 10 6 cells mL, and maintained as described above.
On the day of peptone addition, the cells were centrifuged and transferred to 5 ml fresh medium (RPMI 1640 or LBTC-CDM) containing peptone(s) atmosphere. Cell density and viability were assessed by the trypan blue dye exclusion method using a haemocytometer. The PCV was determined as described above.

Results
The This wheat-derived peptone has a remarkably higher content of glutamate (29 g/L) and proline (9 g/L) when compared to the other peptones, while the concentration of aspartate was the lowest (2.9 g/L) (see supplementary material Table ST1).    (Fig. 4). Soy bean-derived Pep 5 was also very effective in all the two-peptone mixtures at 4 g/L (Fig. 4). The effect of 2-peptone mixtures added on day 0 or day 2 at 4 g/L (data presented in figure 4) was expressed as fold variation over control and compared to the averaged effect of the single individual peptones addition (data reported in Fig.   2). The results were grouped by each category of five peptone mixtures (casein + casein, soy + soy, casein + wheat, soy + casein, soy + wheat) and plotted according to the day of feeding (day 0 or day 2). Fig 5 shows a dot plot of the data obtained.
The variation in IgG production obtained with the  Table 1 and Fig. 4). The six mixtures containing peptones derived from casein did not induce an improvement in recombinant protein production ( Fig. 4). All the casein-derived peptones have higher total concentration of some amino acids compared to plant-derived peptones (Lys, Thr, Met, Leu, Ile, Val: see supplementary Table ST1). Pep 6 has the highest free tyrosine and histidine content (65.6% and 41.9%, see supplementary Table ST2) compared to all other peptones. Furthermore, Pep 6 has a high average molecular weight with almost 30 % of the total having a size between 1 and 10 kDa, the highest among the casein-derived peptones tested (see Table 1). In two-peptone mixtures, this  Table ST2). Whether these features have a direct impact on CHO cells productivity remains to be elucidated.  peptone feeds on productivity cannot be easily predicted by testing the peptones individually. The time of feeding also plays an important role. In the present study, we considered the effect of peptone feeding on volumetric productivity, which may result either from increased specific productivity or from increased biomass, or even a combination of the two effects.

Discussion
In a recent report, a proteomic approach was applied to identify in CHO cells intracellular protein with either induced or suppressed expression upon peptones feeding 26  Also, a new insight has been included to amino acid profile of peptones as feeding strategies of mammalian cell cultures which would hopefully lead to predictable process development strategies.
The results reported here show that a simple feeding strategy with protein hydrolysates was allowed to improve r-protein titers in a CHO cell clone cultivated in a basal serum-free medium.
Furthermore, the two-peptone feeding strategy tested here may give unpredictable results, not always corresponding to the effects of the single peptones fed individually. Peptone concentration and time of addition play a critical role in the feeding strategies. Our high-throughput system, the TubeSpin ® bioreactor 50 system based on orbital shaking, proved again to be an efficient tool for cell culture optimization 27; 28 . The data presented in this study suggest that cell-line tailored feeding strategy based on peptone feeds can be developed which allow improving the productivity of recombinant CHO cell lines in basal media.